Vladimir Yarov-Yarovoy - US grants

DESCRIPTION (provided by applicant): This proposal is a request for a Mentored Research Scientist Development Award (K01) for senior fellow researcher Dr. Vladimir Yarov-Yarovoy. The candidate holds advanced degrees in Physics (M.S.) and Biochemistry and Molecular Biology (Ph.D.) and is well trained in the field of Neuroscience. The candidate's ultimate goal is to lead an independent research group, which will conduct fundamental research in the field of neuroinformatics - the interdisciplinary field that bridges research in neuroscience with that in informatics. Successful completion of this proposal will further enhance the candidate's expertise in the field of neuroscience and also will allow him to develop the essential theoretical knowledge and experience in the field of informatics. The candidate's long-term research interests include structure function studies of neuronal ion channels and computational modeling of protein structure, protein-protein, and protein-drug interactions. Dr. Yarov-Yarovoy's training will consist of computational biology and computer programming courses and research work under the dual mentorship of Drs. William Catterall and David Baker. Dr. Catterall is an accomplished investigator in studying the structure and function of the brain voltage-gated sodium and calcium channels. Dr. Baker is an accomplished investigator in predicting the three-dimensional protein structure using novel computational methods. The goal of the project is to develop three-dimensional models of the brain voltage-gated sodium and calcium channels. These models will provide key structural information on the molecular basis of the brain sodium and calcium channel gating, their modulation by second messenger-activated protein phosphorylation and by peptide neurotoxins, and their interaction with therapeutically useful pore-blocking drugs. The novel features of the models will be experimentally tested using site-directed mutagenesis and electrophysiological techniques. The novel computational tools developed in this project will be generalized for structure prediction of other ion channels. Finally, understanding the brain sodium and calcium channel function and modulation on the structural level will lead to a better understanding of mechanisms of neuromodulation, which in turn will give us profound insights into the fundamental mechanisms underlying learning, memory, emotion and behavior.

Project Summary Capsaicin- and heat-activated TRPV1 ion channel is a primary nociceptor for both chemical and thermal stimuli, hence an attractive target for pain medication. Despite the availability of cryo-EM structures of TRPV1 at up-to-2.9 Å resolutions, molecular mechanisms underlying TRPV1 activation remains unclear. A major limitation for obtaining mechanistic information from the cryo-EM structures is the general lack of resolution to determine side-chain orientation and the associated atomic interaction. We recently demonstrated that the limitation could be overcome by combining Rosetta structural prediction with site-specific functional tests such as thermodynamic mutant cycle analysis that serve to constrain, validate, and improve structure prediction. Using this iterative approach, in combination with molecular dynamics (MD) simulation and site-specific fluorescence recordings including FRET and patch fluorometry, our proposed study aims to identify functional interactions in the capsaicin-binding domain and the outer pore and, more importantly, to reveal dynamic changes of these interactions during capsaicin- and heat-induced activation. We will take particular advantage of our newly designed fluorescent capsaicin analogs that allow us to directly monitor ligand binding, as well as the fluorescent unnatural amino acid (FUAA) incorporation method to introduce a small fluorophore to the channel's moving parts. Our goal is to elucidate key molecular interactions that mediate chemical and thermal activation, thus providing a molecular framework to guide pharmaceutical intervention.

Project Summary Capsaicin- and heat-activated TRPV1 ion channel is a primary nociceptor for both chemical and thermal stimuli, hence an attractive target for pain medication. Despite the availability of cryo-EM structures of TRPV1 at up-to-2.9 Å resolutions, molecular mechanisms underlying TRPV1 activation remains unclear. A major limitation for obtaining mechanistic information from the cryo-EM structures is the general lack of resolution to determine side-chain orientation and the associated atomic interaction. We recently demonstrated that the limitation could be overcome by combining Rosetta structural prediction with site-specific functional tests such as thermodynamic mutant cycle analysis that serve to constrain, validate, and improve structure prediction. Using this iterative approach, in combination with site-specific fluorescence recordings including FRET and patch fluorometry, our study aims to identify functional interactions in the capsaicin-binding domain and the outer pore and, more importantly, to reveal dynamic changes of these interactions during capsaicin- and heat-induced activation. We will take particular advantage of the fluorescent unnatural amino acid (FUAA) incorporation method to introduce a small fluorophore to the channel?s moving parts. Our goal is to elucidate structural mechanisms for key molecular interactions that mediate chemical and thermal activation, thus providing a molecular framework to guide pharmaceutical intervention.

Project Summary Heat- and capsaicin-activated ion channel TRPV1 is an important cellular sensor for maintaining normal body temperature and detecting noxious ambient environment, hence an important drug target. TRPV1 also serves as a prototypical model for understanding polymodal activation of diverse TRP channels. Structures of TRPV1 have been solved by cryo-electron microscopy (cyro-EM) at up-to-2.9- Å resolutions, yet mechanistic understanding of channel activation, in particular by heat, remains preliminary. A major limitation has been the lack of effective tools for probing structural dynamics in functional channels. We have recently identified two structurally related peptide toxins from the venom of Chinese red-headed centipede, termed RhTx1 and RhTx2, that exhibit highly specific and sub-?M affinity binding to the outer pore region of TRPV1. Interestingly, while RhTx1 strongly activates the channel, RhTx2, having just four additional amino acids at its N-terminus, is a potent inhibitor. Our preliminary study further suggested that RhTx1 specifically promotes heat activation. Because these small, compact peptide toxins can be readily synthesized and modified, they offer unique opportunities to probe the molecular mechanism underlying TRPV1 activation, with their rapid ON and OFF kinetics being particularly favorable for biophysical investigation. The proposed study will take advantage of these novel toxins to gain a better understanding of TRPV1 activation mechanism by revealing its structural dynamics using a multidisciplinary approach combines structural, electrophysiological, optical, and computational modeling methods.